Carbonic anhydrases (CAs) are a large family of zinc metalloenzymes that catalyze the reversible hydration of carbon dioxide. They participate in a variety of biological processes, including respiration, calcification, acid-base balance, bone resorption, and the formation of aqueous humor, cerebrospinal fluid, saliva, and gastric acid. They show extensive diversity in tissue distribution and in their subcellular localization. CAIX (also known as MN) is a transmembrane protein encoded by the CA9 gene and has been shown to be expressed in cancer tumors.
Tumor Microenvironment
The microenvironment of solid tumors is shaped by a biochemical and physiological interplay between cancer cells on one hand, and supporting stromal and vessel cells on the other hand. Tumors often grow at a rate that exceeds the blood supply capacity of the host vasculature, which would normally impede further development of most normal tissues. Tumors, however, escape this negative feedback and maintain high respiratory rates and are surviving even when oxygen, a principal blood-borne substrate, is scarce. Indeed, low oxygen tension (hypoxia) is a cardinal feature of the tumor milieu (Gatenby and Gillies (2004) Nat Rev Cancer 4: 891-899). Hypoxia is not only a consequence of tumor respiration, but also a trigger for an altered program of gene expression, featuring hypoxia-inducible genes (Gleadle and Ratcliffe (1998) Mol Med Today 4: 122-129; Harris (2002) Nat Rev Cancer 2:38-47), that is involved in progressing cancer towards a more aggressive disease phenotype (Fang et al., (2008) Semin Cancer Biol 18: 330-337). Many of the hypoxia-regulated genes are controlled by hypoxia-inducible factor, a transcription factor that is otherwise inactivated in the presence of oxygen. The targets of hypoxia-inducible factor include genes that encode proteins involved in glucose metabolism, blood vessel growth, oxygen carriage, iron metabolism and numerous other processes.
CAIX Expression and Function
CAIX was originally cloned in the mid 1990s by Pastorek and coworkers (Pastorek et al., (1994) Oncogene 9: 2877-2888). CAIX is a hypoxia-inducible enzyme and a component of the pH regulatory system invoked by cells to combat the deleterious effects of a high rate of glycolytic metabolism (Wykoff et al., (2000) Cancer Res 60: 7075-7083).
CAIX expression, unlike the expression of most other CA isoforms, is associated with many tumors (Pastorek et al., (1994) supra; De Simone and Supuran, (2010) Biochim Biophys Acta 1804: 404-409). Indeed, very few normal tissues (with the notable exception of stomach (Pastorekova et al., (1997) Gastroenterology 112: 398-408)) express significant levels of CAIX, so positive staining for CAIX is now an established marker of tumor hypoxia and a clinical indicator of aggressive cancers (for example breast and bone cancer) with a poor prognosis (reviewed in McDonald et al (2012) Oncotarget 3:84-97).
CAIX functions to help produce and maintain an intracellular pH (pHi) favorable for tumor cell growth and survival, while at the same time participating in the generation of an increasingly acidic extracellular space, facilitating tumor cell invasiveness.
CAIX is membrane tethered and its catalytic domain faces the extracellular environment. The crystal structure of CAIX has been resolved, showing that CAIX exists as a dimer (Alterio et al., (2009) Proc Natl Acad Sci USA 106: 16233-16238; De Simone and Supuran, (2010) supra). The protein contains a proteoglycan-like domain and an intracellular carboxy terminal tail that may be involved in cell-cell adhesion and in regulating the catalytic process. Recent work (Innocenti et al., (2009) Bioorg Med Chem Lett 19: 5825-5828) has proposed that the presence of the proteoglycan domain, rich in acidic amino acid residues, reduces the inhibitory effects of H+ ions on CAIX activity. This is observed as a shift in the pH sensitivity of CAIX activity by half a pH unit towards more acidic values, enabling CAIX to remain catalytically active in the acidic extracellular milieu that is typical of solid tumors.
CAIX and Cancer
CAIX is an especially attractive target for cancer therapy, in part because it is over-expressed in a wide variety of solid tumors, but is expressed in a limited way in normal tissues. In human tissue, strong expression of CAIX is generally limited to the basolateral surface of proliferating crypt enterocytes of the duodenum, jejunum and ileal mucosa (Pastorekova et al (1997) supra; Saarnio et al (1998) J Histochem Cytochem 46:497-504). However, diffuse, weak CAIX expression has also been reported in other tissues.
CAIX is over-expressed in many solid tumors, and there is a well established relationship between the expression of CAIX and patient prognosis. CAIX expression, as detected by immunohistochemical staining of tissue sections, is upregulated and associated with poor prognosis in cancers of the lung, colon, breast, cervix, bladder, kidney, brain, head and neck, and oral cavity (reviewed in McDonald et al (2012) supra). Furthermore, recent studies have examined the expression of CAIX in cohorts of from hundreds to thousands of patients using tissue microarray strategies. Using this high throughput platform, CAIX has been validated as a biomarker of a poor prognosis in breast, lung, ovarian and bladder cancer as well as in astrocytomas (reviewed in McDonald et al (2012), supra).
There is now an increasing focus on the use of CAIX for clinical detection and prognostic evaluation. Initial studies, involving relatively small sample sizes, showed that soluble CAIX was upregulated in serum in patients with solid tumors (Woelber et al (2010) Gynecol Oncol 117:183-188; Hyrsl et al (2009) Neoplasma:56:298-302) and recent studies have shown an association between soluble CAIX and patient prognosis. Preoperative serum CAIX concentrations in vulvar cancer correlate with intratumoral expression, and increased serum CAIX levels are associated with a poor prognosis (Kock et al (2011) Int J Gynecol Cancer 21:141-148). Serum levels of CAIX in metastatic breast cancer were also correlated with a poor prognosis, as well as with the incidence of circulating tumor cells (Muller et al (2011) Breast Cancer Res: 13: R71). Similarly, in NSCLC, high plasma levels of CAIX were associated with significantly shorter overall survival pie et al (2010) Br J Cancer. May 25; 102(11):1627-35).
Pharmacologic interference of CAIX catalytic activity using monoclonal antibodies or CAIX-specific small molecule inhibitors, consequently disrupting pH regulation by cancer cells, has recently been shown to impair primary tumor growth and metastasis.
However, monoclonal antibodies are not always optimal for targeting solid tumors (neither for diagnostic nor for therapeutic pay-load purposes). Therapeutic effect is dependent on an efficient distribution of the drug throughout the tumor, and molecular imaging depends on a high ratio between tumor uptake and surrounding normal tissue.
Since tumor penetration rate (including extravasation) is negatively associated with the size of the molecule, the relatively large antibody molecule inherently has poor tissue distribution and penetration capacity. Moreover, for molecular imaging, the extraordinarily long in vivo half-life of antibodies results in relatively high blood signals and thereby relatively poor tumor-to-blood contrasts.
The continued provision of agents with a high affinity for CAIX remains a matter of substantial interest within the field. Of great interest is also the provision of uses of such molecules in the treatment and diagnosis of disease.